We have studied the pathogenesis of viral hepatitis and the molecular basis for virulence and attenuation of these important pathogens. We have shown previously that virulence and attenuation of hepatitis A virus (HAV) are controlled principally by two genes: VP1/2A and 2C. However, attenuating mutations are strongly selected against in vivo, resulting in the emergence of virulent variants. This has important implications for the development of live attenuated hepatitis A vaccines. The pathogenesis of hepatitis A is also being studied in the chimpanzee model by microarray analysis. Both innate and adaptive responses have been recorded. Interestingly, HAV does not trigger as robust an up-regulation of certain interferon stimulated genes as HCV and HDV infections. This is surprising because HAV and HCV are both single-stranded RNA viruses with a double-stranded replicative form and HDV is a single-stranded virus with extensive base pairing that is perceived as double-stranded RNA. Specifically, in FY 2009, and 2010 we compared the innate and adaptive immune responses to HAV infection in several experimentally infected chimpanzees, including animals that received virulent inocula and those that were infected with attenuated strains of HAV. Animals infected with virulent strains had a robust innate immune response (although not as robust as that seen in HCV-infected chimpanzees), whereas the response was less marked in the attenuated infections. Interestingly, innate immune responses were abbreviated in the latter chimpanzees, but detection of the innate immune response was almost as sensitive a diagnostic test of infection as was detection of specific serologic or molecular probes of HAV infection. The adaptive immune responses were also present, but not as robust as the adaptive immune responses in HCV infections of chimpanzees. It will be important to search for viral mechanisms of control that may block some of these systems. Although rare in the United States, hepatitis E is the single most important cause of acute hepatitis among adults throughout Asia, the Middle East and North Africa. Like most of the hepatitis viruses, it replicates poorly or not at all in cell culture and cannot be transmitted to small laboratory animals. We have developed replicons for the study of HEV in vitro;these tools are permitting a detailed molecular analysis of viral replication that can be confirmed in vivo with molecularly engineered infectious cDNA clones of the virus. In addition, with colleagues, we are developing small animal models (swine HEV in swine, avian HEV in chickens) that, with nonhuman primate models of HEV, provide an unprecedented opportunity for studying the comparative pathogenesis of hepatitis E viruses. Finally, hepatitis E has also been studied by microarray and a brisk innate but weak adaptive immune response has been seen. Specifically, in FY 2009 and 2010, we compared the innate and adaptive immune responses of several chimpanzees to experimental HEV infections with those observed in chimpanzees that had been experimentally infected with HCV or HAV. In comparison with those infections, HEV infections were characterized by an abbreviated and somewhat shortened adaptive immune response. This is particularly interesting in light of other observations that antibody titers tend to diminish more rapidly in HEV infections than in infections with the other hepatitis viruses. More specifically, in 2010, we completed a microarray analysis of HCV and HEV infections of multiple chimpanzees, comparing the sequence of human and chimpanzee genomes for suitability in interpreting Affymetrix microarray data obtained from serial clinical samples of chimpanzees infected with one or the other of the two viruses. We found that the human genome sequence was more sensitive and specific for identifying up-regulated and down-regulated genes, probably because it is better curated than the more recently sequenced chimpanzee genome. We also compared two analytic methods, a method utilizing Affymetrix probe sets and correlation coefficients with one utilizing individual perfectly matched probes and t-test analysis. Both analyses demonstrated similarities and differences in the host immune response to these two RNA viruses. This information will be useful for a better understanding of the pathogenesis of viral hepatitis. In 2011 we completed a study of HEV in rats that we had begun some years earlier. The sequence of the rat hepatitis E virus became available and permitted us to reevaluate extensive transmission studies of rat HEV in laboratory rats. We obtained partial sequence data, prepared pools of known infectivity titer, performed pathogenesis studies and determined cross-species susceptibility of the rat virus. We concluded that the rat HEV does not produce a robust infection in laboratory rats and is not transmissible to nonhuman primates, suggesting that it does not pose a threat to humans. In other collaborative studies we tested sera from patients diagnosed as having drug-induced liver injury (DILI) for evidence of HEV infection and found that 3% of 318 patients actually had hepatitis E. The hepatitis E patients were mostly older men (89% male, mean age 67 years) and two were HIV-positive. We concluded that a small but important proportion of cases of acute liver injury suspected to be drug-induced are actually hepatitis E.